CA2100352A1

CA2100352A1 - Integrin .beta. subunit and uses thereof

Info

Publication number: CA2100352A1
Application number: CA002100352A
Authority: CA
Inventors: Dean Sheppard; Vito Quaranta; Robert Pytela
Original assignee: Individual
Current assignee: Scripps Research Institute; University of California San Diego UCSD
Priority date: 1991-01-11
Filing date: 1991-01-11
Publication date: 1992-07-12
Also published as: WO1992012236A1

Abstract

The present invention provides substantially pure integrins containing a novel .beta. subunit designated as .beta.6. The novel .beta.6 subunit forms heterodimers with .alpha.V and .alpha.F.
Methods of controlling cell adhesion using the .beta.6-containing integrins are also provided.

Description

WO92/12236 ~ v~ PCT/US91/00236 A NOVEL INTEGRIN ~ SUBUNIT
AND USES THEREOF

This work was supported in part by research grants HL/AL 33259, CA-47541 and CA~47858 from the National Institutes of Health. The U.S. Government has rights in the invention.

TECHNICAL FIELD

This invention relates to receptors for adhesion peptides, and more specifically to a novel receptor subunit having affinity for extracellular matrix molecules.

BACKGROUND ART

Multicellular organisms, such as man, have some 1014 cells which can be divided into a minimum of fifty different types, such as blood cells and nerve cells.
During the course of growth and development, cells adhere to other cells, or to extracellular materials, in specific and orderly ways. Such cell adhesion mechanisms appear to be of importance in mediating patterns of cellular growth, migration and differentiation, whereby cells develop specialized characteristics so as to function as, for example, muscle cells or liver cells.
Cell adhesion mechanisms are also implicated in dedifferentiation and invasion, notably where cells lose their specialized forms and become metastasizing cancer cells.

The mechanisms underlying the interactions of cells with one another and with extracellular matrices are not fully understood, but it is thought that they are mediated by cell s~rface receptors which specifically reoo~nize and bind to a cognate ligand on the surface of cells or in the extracellular matrix.

W O 92/12236 PC~r/US91/00236 ,~ 1 iJ ~ J ~ 2 The adhesion of cells to extracellular matrices and their migration on the matrices is mediated in many cases by the binding of a cell surface receptor to an Arg-Gly-Asp containing sequence in the matrix protein, as reviewed in Ruoslahti and Pierschbacher, Science 238:491 (1987). The Arg-Gly-Asp sequence is a cell attachment site at least in fibronectin, vitronectin, fibrinogen von Willibrand, thrombopondin, osteopontin, and possibly various collagens, laminin and tenascin. Despite the similarity of their cell attachment sites, these proteins can be recognized individually by their interactions with specific receptors.

The integrins are a large family of cell surface glycoproteins that mediate cell-to-cell and cell-to-matrix adhesion as described, for example, in the Ruoslahti and Pierschbacher article cited above. All known members of this family of adhesion receptors are heterodimers consisting of an ~ and a B subunit noncovalently bound to each other. When the integrin family was first identified, integrins were grouped into three subfamilies based on the three B subunits that were initially recognized (B1, B2 and B3). Over the past few years, the primary structures of three integrin B
subunits from mammalian cells and one from Drosophila have been deduced from cDNA.

Each ~ subunit was thought to associate uniquely with a single B subunit. Eleven distinct ~
subunits have thus far been described. As new integrins have been identified, however, it has become clear that this grouping is not entirely satisfactory, since there are clearly more than three B subunits and since some subunits can associate with more than one B subunit as described, for example, in Sonnenberg et al., J. Biol.
Chem. 265:14030-14038 (1988).

WO92/12236 ~ PCT/US91/00236 ~ J ,~

Because of the importance of integrins in mediating critical aspects of both normal and abnormal cell processes, a need exists to identify and characterize different integrins. The present invention satisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

The present invention relates to a substantially purified ~ subunit of an integrin cell surface receptor designated as B6. The amino acid sequence of ~6 is provided in Figure 3.

The present invention also relates to amino acid fragments specific to B6 that have a variety of uses.
The invention further relates to vectors having a gene encoding such fragments. Host cells containing such vectors are also provided. The nucleic acids encoding ~6 as well as nucleic acids that specifically hybridize with the nucleic acids encoding ~6 sequences are other aspects of the present invention.

In a further aspect, the present invention relates to a substantially purified integrin comprising B6 bound to an ~ subunit, particularly ~v or ~F. Methods of blocking the attachment of the B6-containing integrins to its ligand and of detecting the binding of such integrins to its ligand are also provided.

The present invention also relates to methods of increasing or decreasing cell adhesion in cells expressing a B6-containing integrin by overexpressing the integrin or by binding the integrin with a ligand, such as vitronectin.

WO92/12236 ~ J ~ PCT/US91/~236 BRIEF DESCRIPTION OF THE FIGURES

Figure l shows the design of PCR primers.

Figure 2 shows a map of sequencing strategy.

Figure 3 shows the nucleotide sequence and amino acid translation for human (H) and guinea pig (GP) B6 ~

Figure 4 shows the alignment of B6 with four previously reported integrin B subunits.

Figure 5 shows the alignment of partial nucleotide and amino acid sequences from human (H) and guinea pig (GP) B1, B2, B3, and B6 for the region just downstream from the B3F primer.

D~TAILED DESCRIPTION OF THE INVENTION

The present invention provides a composition of matter relating to a novel, substantially purified integrin B subunit, referred to herein as B6. The amino acid sequence of B6 is also provided and is shown in Figure 3.

By "substantially purified" is meant substantially free of contaminants normally associated with a native or natural environment.

By "B6" is meant a polypeptide having substantially the same amino acid sequence and binding functions of the polypeptides encoded by the sequences set forth in Figure 3 for human and guinea pig B6. Thus, modified amino acid sequences that do not substantially destroy the functions and retain the essential sequence of B6 are included within the definition of B6. Amino WO92/12236 ~ l V~v~ PCT/US91/00236 acid sequences, such as the sequence for B1, B2 and ~3, having less than 50% homology with the sequence of B6, are not substantially the same sequence and, therefore, do not fall within the definition of B6. Given the amino acid sequences set forth herein, additions, deletions or substitutions can be made and tested to determine their effect on the function of B6. In addition, one skilled in the art would recognize that certain amino acids, such as the conserved cystines, for example, can be modified to alter a binding function of B6.

Amino acids are identified herein by the standard one-letter abbreviations, as follows:

Amino Acid Symbol Alanine A
Asparagine N
Aspartic acid D
Arginine R
Cysteine C
Glutamine Q
Glutamic acid E
Glycine G
Histidine H
Isoleucine Leucine L
Lysine K
Methionine M
Phenylalanine F
Proline P
Serine S
Threonine T
Tryptophan Tyrosine y Valine V

Based on its amino acid sequence, the B subunit of the present invention is clearly different from B1, B2 B3 and other B subunits that have recently been discovered. For example, the ll-amino acid carboxyl-terminal extension on B6 distinguishes it from B1~ B2, and WO92/12236 ~ ~ PCT/US91/00236 B3. The short cytoplasmic tails of B~, Bz, and B3 arethought to be sites of interaction with the cytoskeleton and regions for the transduction of signals initiated by interactions of the large extracellular domains with ligands. These cytoplasmic tails may also be targets for regulation of integrin function. The distinctive 11-amino acid cytoplasmic tail of B6 indicates that its regulation or pathways for signal transduction may be different from those of Bt, B2 and B3.

In addition to B1, B2 and B3, recent studies have suggested the existence of as many as five other integrin B subunits. A B subunit with a molecular weight of approximately 210,000 (B4) has been found associated with the integrin subunit 1'6" in colon carcinoma cells and in a variety of other tumor cells of epithelial origin as described, for example, in Kajiji et al., EMBO
J., 8:673-680 (1989). On the basis of its high molecular weight, 210,000 compared with the predicted size of 106,000 of the subject novel protein, and on the basis of its clearly different amino-terminal sequence, it is apparent that B4 is not the same as the subject polypeptide.

Another B subunit, originally called Bx was identified in epithelial-derived tumor cells in association with the integrin subunit v as described, for example, in Cheresh et al., Cell 57:59-69 (1989).
This B subunit, having a distinctive amino-terminal sequence, was recently renamed B5. Based on recent studies of purified preparations, Bs clearly differs from the B subunit of the present invention. Because the B
subunit described in the present report is distinct from each of the five B subunits for which sequence information is available, it has been designated as B6.

The existence of two other integrin B subunits WO92/12236 ~ v t PCT/US91/00236 has been inferred from the identification of unique proteins after immunoprecipitation o~ surface-labeled cell lysates with antibodies to known ~ subunits. One of these novel proteins, called Bs was found in association with ~v in the human osteosarcoma cell line MG-63, in the fibroblast cell line AFl523, and in human endothelial cells as described, for example, in Freed et al., EMBO J.
8:2955-2965 (1989). This subunit is also different from B6 since Bs is expressed in MG-63 cells while B6 is not expressed in these cells as shown in Table 1.

The other novel integrin B subunit identified by co-immunoprecipitation of known ~ subunits, Bp, is a protein of about Mr 95,000 that is found to be associated with ~4, an ~ subunit first found as part of the lymphocyte homing receptor VLA-4 as described, for example, in Holzmann et al., Cell 45:37~46 (1989). This subunit is also distinct from B6 since Bp is expressed in lymphocytes while B6 is not expressed in lymphocytes as shown in Table 1.

W092/12236 ~ PCT/US91/00236 Distribution of B6 ~YE~ Results Source Cell Lines:
5 FG-2 Pancreatic + Kajiji et al.
EMBO J 3:673-80 (1989) Panc I Pancreatic - Dr. Metzgar, Duke U., N.C.
Colo-396 Colon CA + Dr. L. Walker, Cytel, San Diego, CA
UCLA P3 Lung CA + Dr. L. Walker, Cytel, San Diego, CA
Hela Cervical - ATCC #CCL-2 10 Jar Chorio CA + ATCC #HTB 36 HT 1080 Fibrosarcoma - ATCC #CCL 121 U 937 Monocytoid - ATCC #CRL 1593 M 21 Melanoma - Dr. R.
Reisfeld, Scripps Clinic & Research Foundation, La Jolla, CA
B 16 Melanoma - Dr. R.
Reisfeld - Scripps Clinic & Research Foundation, La Jolla, CA
15 MG 63 Osteosarcoma - ATCC #CRL 1427 Tissues:
Cervix +
Aortic Endothelium Leukocytes ~91 U U ~
The invention also provides an integrin comprising B6 bound to an ~ subunit. B6, consistent with recent findings of other B subunits, can associate with a variety of ~ subunits to form a functional integrin. In one embodiment, B6 associates with ~v In another embodiment, B6 associates with another ~ subunit referred to herein as ~F. The v B6 integrin, as well as other integrins containing B6, can bind molecules, for example extracellular matrix molecules. Such molecules are referred to herein as ligands. In a specific embodiment, certain B6-containing integrins can bind Arg-Gly-Asp-containing polypeptides such as vitronectin or fibronectin. The binding of B6-containing integrins to various ligands can be determined according to procedures known in the art and as described for example, in Ruoslahti & Pierschbacher, Science 238:491-497 (1987).

The invention also provides an amino acid fragment specific to B6. Since B6 is a novel molecule, it contains many fragments which are specific for this B
subunit. Fragments specific to B6 contain se~uences having less than 50% homology with sequences of other known integrin B subunit fragments. These fragments are necessarily of sufficient length to be distinguishable from known fragments and, therefore, are "specific for 2S B6." The amino acid sequence of such fragments can readily be determined by referring to the figures which identify the B6 amino acid sequences. These fragments also retain the binding function of the B6 subunit and can therefore be used, for example, as immunogens to prepare reagents specific for B6 or as an indicator to detect the novel B6-containing integrin of the present invention.
One skilled in the art would know of other uses for such fragments.

The invention also provides a reagent having specificity for an amino acid sequence specific for B6.

WO92/12236 ~ J ~ PCT/US91/00236 Since ~6 is a novel protein with at least 50% amino acid differences over related B subunits, one skilled in the art could readily make reagents, such as antibodies, which are specifically reactive with amino acid sequences specific for B6 and thereby immunologically distinguish B6 from other molecules. Various methods of making such antibodies are well established and are described, for example, in Antibodies, A Laboratory Manual, E. Harlow and D. Lane, Cold Spring Harbor Laboratory 1988, pp. 139-283 and Huse et al., Science 24:1275-1280 (1988).

The invention also provides nucleic acids which encode B6. Examples of such sequences are set forth in Figure 3. Following standard methods as described, for example, in Maniatis et al., Molecular Clonina, Cold Spring Harbor (1982), nucleic acid sequences can be cloned into the appropriate expression vector. The vector can then be inserted into a host, which will then be capable of expressing recombinant proteins. Thus, the invention also relates to vectors containing nucleic acids encoding such sequences and to hosts containing these vectors.

The sequences set forth in Figure 3 also provide nucleic acids that can be used as probes for diagnostic purpos0s. Such nucleic acids can hybridize with a nucleic acid having a nucleotide sequence specific for B6 but do not hybridize with nucleic acids encoding non-B6 proteins, particularly other cell surface receptors. These nucleic acids can readily be determined from the sequence of B6 and synthesized using a standard nucleic acid synthesizer. Nucleic acids are also provided which specifically hybridize to either the coding pr non-coding DNA of B6.

Integrin cell surface receptors bind ligands, such as extracellular matrix molecules. However, the WO92~12236 ~ ~ ~ PCT/US91/~236 binding of the integrin to the ligand can be blocked by various means. For example, the binding of a B6-containing integrin can be blocked by a reagent that binds the ~6 subunit or the B6-containing integrin.
Examples of such reagents include, for example, Arg-Gly-Asp-containing peptides and polypeptides, ligand fragments containing the integrin binding site, as well as antibodies specifically reactive with B6 or a B6-containing integrin. Alternatively, the blocking can be carried out by binding the ligand or fragment thereof, recognized by a ~6-containing integrin with a reagent specific for the ligand at a site that inhibits the ligand from binding with the integrin. Since the binding of a B6-containing integrin to its ligand can mediate cell adhesion to an extracellular matrix molecule, preventing this binding can prevent cell adhesion. Alternatively, cell adhesion can be promoted by increasing the expression of B6-containing integrins by a cell.

Finally, the invention provides a method of detecting ligands which bind a B6-containing integrin.
The method comprises contacting a B6-containing integrin with a solution containing ligands suspected of binding B6-containing integrins. The presence of ligands which bind a B6-containing integrin is then detected.

In summary, the invention claims:

1. A substantially purified integrin cell surface receptor subunit comprising B6.

2. The substantially purified integrin cell surface receptor subunit of claim 1 having the amino acid sequence set forth in Figure 3 for human.

3. A substantially purified integrin comprising B6 bound to an ~ subunit.

) 12 ,_ lU~J j~

4. The integrin of claim 3, wherein the subunit is ~v

5. The integrin of cl2im 3, wherein the subunit is ~F.

6. A substantially purified amino acid fragment specific to B6.

7. A vector comprising a gene encoding for the amino acid fragment of claim 6.

8. A host ~ontaining the vector of claim 7.

9. A reagent having specificity for an amino acid sequence specific for B6.

10. The reagent of claim 9, wherein the reagent is an antibody.

11. A substantially purified nucleic acid encoding B6.

12. A substantially purified nucleic acid which specifically hybridizes with a nucleotide sequence of the nucleic acid of claim 11.

13. A substantially purified nucleic acid which specifically hybridizes with the nucleic acid of claim 12 and does not hybridize with a nucleic acid encoding a non-B6 polypeptide.

W092/t2236 ~ 1 U~ PCT/US91/00236

14. A method of preventing the binding of a cell expressing a B6-containing integrin to ligand capable of binding to said B6-containing integrin, comprising blocking the binding of the B6-containing integrin and the ligand.

15. The method of claim 14, wherein the blocking is effected by binding the B6-containing integrin with a reagent specific thereto.

16. The method of claim 14, wherein the blocking is effected by binding the ligand of the B6-containing integrin with a reagent specific for the ligand.

17. The method of claim 15, wherein the reagent is an RGD-containing peptide or polypeptide.

18. The method of claim 15, wherein the reagent is a ligand fragment containing an integrin binding site.

19. A method of detecting a ligand that binds a B6-containing integrin, comprising contacting the B6-containing integrin with a solution containing the ligandsuspected of binding B6-containing integrins and detecting the presence of the ligand bound to the B6-containing integrin.

20. A method of increasing cell adhesion in cells expressing a B6-containing integrin, comprising overexpressing the B6-containing integrin in a cell.

21. A method of decreasing cell adhesion in cells expressing a B6-containing integrin comprising binding the B6-containing integrin with a ligand.

WO92/12236 i UU ~ ~ PCT/US91/00236 The following examples are intended to illustrate but not limit the invention.

EXAMPLE I
Identification of a Novel ~ Subunit Generation of cDNA Fraoments by Polymerase Chain Reaction Tracheal epithelial cells harvested from male Hartley outbred guinea pigs (Charles River Breeding Laboratories, Bar Harbor, ME) were grown to confluence over 10-14 days on collagen-impregnated microporous filters commercially available from Costar. RNA was harvested from thPse primary cultures, and mRNA was purified over oligo(dT)-cellulose columns using the Fast Track mRNA isolation kit (Invitrogen, San Diego, California). Two to 5 ~g of mRNA was used as a template for cDNA synthesis catalyzed by 200 units of Moloney murine leukemia virus reverse transcriptase (Bethesda Research Laboratories, Gaithersburg, MD) in a 20-40 ~l reaction volume. One to 5 ~l of the resultant cDNA was used as a template for polymerase chain reaction (PCR).
PCR was carried out in a reaction volume of 25-200 ~l.
In addition to the template cDNA, each PCR reaction contained 50 mM KCl, l0 mM Tris-HCl (pH 9.0 at 25-C), 1.5 mM MgCl2, 0.01% gelatin, 0.1% Triton X-lO0, 0.2 mM each of dATP, dGTP, dCTP and dTTP, and 0.05 units/~l Taq DNA
polymerase (obtained from either United States Biochemical Corporation, Cleveland, OH, or from Promega, Madison, WI).

For each reaction, two oligonucleotide primers were also added to obtain a final concentration of l ~M
each. The primer pairs are identified below. Each reaction mixture was overlaid with mineral oil, heated to 95~C for 4 min. in a thermal cycler (Ericomp, San Diego, CA), and then subjected to 30 cycles of PCR. Each cycle O92/12236 ~ PCT/US91/00236 l V l~ J w consisted of 45 seconds at 95C, 45 seconds at 53C, and l min. at 72C. Immediately after the last cycle, the sample was maintained at 72C for l0 min.

The results of each PCR reaction were analyzed by gel electrophoresis in l.5% agarose. Reactions that produced fragments of the expected size were electrophoresed in l.5% low gel temperature agarose (Bio-Rad Laboratories, Richmond, CA). The appropriate size band was excised, melted at 68C, and the DNA was purified by extraction with phenol/chloroform and precipitation in ethanol and ammonium acetate.

PCR Primers To obtain the initial fragment of the novel B
subunit cDNA described herein, degenerate mixtures of PCR
primers were used. Oligonucleotides were synthesized, trityl-on, by the Uni~ersity of California, San Francisco Biomolecular Resource Center using a DNA synthesizer with standard procedures, and purified over Nen-sorb cartridges (DuPont-New England Nuclear, Boston, M~).
These consensus primer mixtures were designed to anneal with the nucleotides encoding the highly conserved sequence Asp-Leu-Tyr-Tyr-Leu-Met-Asp-Leu (primer BlF) and Glu-Gly-Gly-Asp-Ala-Ile-Met-Gln (primer B2R) that flank an approximately 300-nucleotide region beginning approximately 130 amino acids from the amino terminus of each of the integrin B subunits sequenced to date. The sequences of the primers identified herein are depicted in Figure l.

On the basis of the initial sequence obtained, a specific forward primer was designed to anneal with the sequence encoding the amino acids Pro-Leu-Thr-Asn-Asp-Ala-Glu-Arg (primer BTE2F) ending approximately 49 W O 92tt2236 PC~r/US91/00236 ~ 16 nucleotides from the 3' end of the region we had sequenced. We also designed an additional forward primer (B3F) and two reverse primers (B3R and B4R) to recognize highly conserved consensus regions encoding the sequences Gly-Glu-Cys-Val-Cys-Gly-Gln-Cys (B3 region) and Ile-Gly-Leu-Ala-heu-Leu-Leu-Ile-Trp-Lys (B4 region). The alignment of these primers with previously published sequences of human Bl, B2 and B3 and chicken Bl is shown in Fig. 1. PCR as described above was performed with cDNA
from guinea pig tracheal epithelial cells and the primer pairs BTE2F/B3R and B3F/B4R.

The primer pair BTE2F/B3R yielded 1095 additional base pairs of new sequence. Based on this sequence another specific primer (BTE3F) was designed to recognize the sequence Val-Ser-Glu-Asp-Gly-Val near the 3' end of this sequence, and PCR was performed with this primer in combination with primer B4R.

Figure 1 shows the design of PCR primers. B
subunit consensus primer mixtures were designed on the basis of alignment of published sequences of human B1, Bz, B3 and chicken Bl. For forward primers (BlF and B3F), the primer sequences included a single nucleotide whenever possible for each of the first two nucleotides of each codon and were usually either degenerate or included deoxyinosine for the third base in codons for amino acids other than methionine. Reverse primers (B2R, B3R, and B4R) were designed in the same manner for the complementary DNA strand. Two specific forward primers were designed to recognize B6. The first (BTE2F) was designed to work across species and was thus degenerate or included deoxyinosine in the third codon position.
The second, BTE3F, was not degenerate and was designed to only recognize guinea pig B6.

W O 92/12236 ~ ~ iJ ~ PC~r/US91/00236 Clonincl of Fraaments Obtained by PCR

Individual fragments were cloned in pBluescript (Stratagene, San Diego, CA) as follows. Purified fragments were resuspended in distilled water containing deoxynucleotides and treated with 2.5 unlts of DNA
polymerase I, large fragment (Promega) to fill in any 3' recessed ends left after the last cycle of PCR. The 5' ends were phosphorylated with 5 units of T4 polynucleotide kinase (New England Biolabs, Beverly, MA).
An aliquot of the above reaction mixture containing approximately 100-200 ng of DNA, was ligated into pBluescript that had been cut with EcoRV (Promega) and dephosphorylated with calf intestinal alkaline phosphatase (Boehringer Mannheim, Indianapolis, IN).
Ligations were performed at 22C for 1 hour with T4 DNA
ligase (Bethesda Research Laboratories). The ligation mixture was used to transform competent Escherichia coli (JM109, Clontech, San Francisco, California). Plasmids containing inserts were purified using the Pharmacia miniprep lysis kit (Pharmacia LKB Biotechnology, Inc., Piscataway, NJ) denatured in 0.3 M NaOH, further purified over spin columns containing Sephacryl S-400 (Pharmacia), and then sequenced using the SequenaseTH version 2.0 sequencing kit (United States Biochemical Corp., Cleveland, OH) and [35S]dATP (Amersham Corp., Arlington Heights ! IL).

Librarv Screeninq PCR fragments generated with the primer pairs BlF/B2R and BTE3F/B4R were uniformly labeled with alpha-[32P]dCTP and used as probes to screen a random-primed cDNA library and an oligo-dT-primed cDNA library both constructed in the plasmid pTZ18R-BstXI (Invitrogen) from mRNA obtained from the human pancreatic carcinoma cell line FG-2. Plasmid was purified from clones found to W092/12236 ~ PCT/US91t~236 I ~J U ~

hybridize with either region, and inserts were sequenced.
A portion of insert DNA from one clone was in turn labeled and used to screen the same libraries. Fourteen independent overlapping clones were sequenced from both ends using primers that recognize regions of the pTZ
polylinker. The regions flanking the 3' end of the putative translated region of the new B subunit were sequenced in both directions from three clones using primers constructed to recognize sequences close to the 3' end. On the basis of the initial sequences thus obtained, an additional internal sequence was obtained from clones T10, Tll, T12 and T14 (Fig. 2) after digestion with specific restriction endonucleases and relegation. Three internal fragments thus generated were subcloned into pBluescript and were also sequenced in both directions. Approximately 90% of the new sequence reported was obtained from both strands of DNA, and 97%
was obtained from two or more overlapping clones (Fig.
2).

Figure 2 shows a map of the sequencing strategy. Shown are the location of clones used to obtain the partial cDNA sequence of gulnea pig B6 (clones lF, 3L, 3N and 3Y, top) and the complete sequence of human B6 (clones Tl-Tl9 bottom). Also shown is the location of the translated region (Protein). The location of the transmembrane domain is shown by the letters TM. Clones shown often represent one of several identical clones. Internal sequence of clones with long inserts was obtained by restriction endonuclease digestion and relegation and by ligation of internal fragments into pBluescript. Specific restriction sites employed are shown (Hind, HindIII; Hinc, HincII; Kpn, KpnI; Pst, PstI). The direction and extent of sequencing are shown by arrows. 1109 and 1110 are the sites recognized by oligonucleotide sequencing primers. Tl8 and Tl9 each terminated in a poly(A) tail. The regions WO92/12236 ~ PCT/US91/00236 ~1 U U ~J~

recognized by the degenerate PCR primers BlF (B1), B2R
(B2), B3R/F (B3)., and B4R (B4) and the B6 primers BTE2F
(BTE2) and BTE3F (BTE3) are noted above the guinea pig cDNA map, kb, kilobases.

Nucleotide Sequence of a Novel Guinea Pig Integrin B
Subunit PCR using cDNA from guinea pig airway epithelial cells and the consensus primer mixtures BlF
and B2R (Fig. 1) amplified DNA fragments with the expected cize of approximately 350 nucleotides. When the fragment DNA was sequenced after cloning into pBluescript, recombinant clones each contained inserts with one of two distinct sequences. One sequence encoded a stretch of 98 amino acids that was 97% identical to the expected region of human B1 and was therefore presumed to be guinea pig Bl. The other sequence encoded 98 amino acids that were only 53~ identical to human B1, 45%
identical to human B2, and 57% identical to human B3 (Fig.
2, clone lF). Both of the guinea pig sequences included the integrin B subunit consensus sequences Ser-X-Ser-Met-X-Asp-Asp-Leu and Gly-Phe-Gly-Ser-Phe-Val, and both contained the 2 cysteine residues found in this region in all known integrin B subunits. These data suggest that one of the two sequences we obtained encoded a new member of the integrin B subunit family.

This novel sequence was extended by further PCR
steps utilizing primers specific for the novel sequence (BTE2F, BTE3F) in combination with two additional degenerate primers (B3R and B4R, see Figs. 1, 2 and 4).
With the primer pair BTE2F/B3R two different cDNA
products were obtained (3L and 3N in Fig. 2) due to an unexpected hybridization of the B3R primer with a site 220 nucleotides further downstream (B3' in Fig. 2). The 1732-nucleotide sequence determined from these clones is O92/12236 PCT/US91/~236 ~ 1 IJ ~ 2 0 shown in Fig. 3.

Figure 3 shows Nucleotide sequence and amino acid translation for human (H) and guinea pig (GP) B6.
The amino acid translation is denoted by the single letter code beneath the second nucleotide of each codon from the translated region of human B6. For the guinea pig sequence, only amino acids that differ from the human sequence are shown. The numbers along the right-hand margin denote the nucleotide or amino acid number of the last entry on each line. The numbering system used starts with the first nucleotide or amino acid available for each sequence shown. The nine potential sites for N-glycosylation in the putative extracellular domain of human B6 are underlined.

Nucleotide Sequence of Human B~

Screening of cDNA libraries constructed from the human pancreatic carcinoma cell line FG-2 with guinea pig cDNA probes lF and 3Y (see Fig. 2) and subsequent screening with a probe constructed from a portion of clone T10 (Fig. 2) produced 14 independent positive clones. The two longest clones (T18 and Tl9) extended to the poly(A) tail. A map of these clones, constructed on the basis of sequence information and of the mobility of inserts cut out of these clones in agarose gels is shown in Fig. 2. This map predicts an mRNA of approximately 5 kilobases including at least a 226-nucleotide untranslated region at the 5' end and, a 2364-nucleotide open reading frame, and a 3' untranslated region of approximately 2.5 kilobases. This molecule has been termed integrin B6.

Fig. 3 shows the partial nucleotide and complete amino acid sequences for human B6 (excludinq most of the 3'-untranslated region) and the alignment of the W092/12236 ~l U ~ ~ PCT/US91/00236 1732 nucleotides of sequence obtained from PCR of guinea pig airway epithelial cell cDNA. Of the 577 amino acids deduced from the region sequenced in both species only 36 residues differ; the amino acid sequences are 94%
identical. Furthermore, of the 1732 nucleotides sequenced in both species, 91% are identical. Nine potential glycosylation sites present in the putative extracellular domain of human B6 are shown by underlining.
All seven of these sites that lie within the 577 amino acids obtained for guinea pig B6 are also present in the guinea pig protein. If all of the potential glycosylation sites are occupied with oligosaccharides having an average molecular weight of 2,500, the predicted molecular weight of human B6 would be 106,000.

Comparison of the 788-amino acid sequence deduced from the open reading frame to the three previously sequenced human B subunits and the myospheroid protein of Drosophila is shown in Fig. 4.

Figure 4 shows the aliqnment of B6 with four previously reported integrin B subunits. Previously published sequences for human B1, human B2, human B3, the myospheroid gene product (Bmyo) of Drosophila, and the novel sequence described as (B6) are shown using the single letter amino acid code. The 56 conserved cysteines are noted by * and the 120 other invariant amino acids by = above each line. The transmembrane domain is underlined. The regions used for ~onstructing the consensus B subunit primers BlF (1), B2R (B2), B3F/R
(B3), and B4R (B4) are labeled below the alignment in bold type. The numbers along the right-hand margin denote the number of the last amino acid in each line beginning from the first amino acid of each putative signal sequence.

W O 92/12236 ~ 22 PC~r/US91/00236 There are 179 amino acid residues that are identical in each of the other 1~ subunits and in J36 including 56 conserved cysteine residues. The overall percentage of identical amino acids between B6 and the other human B subunits is 47% for B3, 42% for B~ and 38%
for B2. Human B6 is also 39~ identical to the Drosophila B subunit. Human B~, B2 and B3 and the ~rosophila B
subunit all have cytoplasmic regions consisting of 41 amino acids (beginning after the putative transmembrane domain shown by the underline in Fig. 4). Although B6 contains each of the 10 conserved amino acid residues in this cytoplasmic region it also contains an ll-amino acid extension at the carboxyl terminus. B6 also contains two Arg-Gly-Asp sequences, one at amino acids 514-516 and the other at 594-596. These regions could serve as recognition sites for other ligands of the integrin family.

PCR using the primer pair B3F/B4R (see Fig. 1) amplified fragments of the expected size of approximately 750 nùcleotides. Cloning and seguencing of the fragments did not result in any additional clones containing the novel B subunit sequence but did result in several clones with inserts encoding an amino acid sequence that was 97%
identical to the corresponding region of human B3 and several others encoding an amino acid sequence that was 93% identical to human B1 (Fig. 5). These are presumably the guinea pig homologues of B1 and B3, respectively. The nucleotide seguences of guinea pig and human B1 are 80~
identical, and those of guinea pig and human B3 are 91%
identical.

Figure 5 shows the alignment of partial nucleotide and amino acid sequences from Human (H) and guinea pig (GP) B1, B2, B3, and B6 for the region just downstream from the B3F primer. Amino acid translations denoted by the one-letter code are shown below the second WO92/12236 ~ PCT/US91/00236 U

nucleotide of each codon. For the guinea pig sequences, only amino acids that differ from the human sequences are shown. The numbers shown along the right-hand margin denote the nucleotide number for human B6. The sequences for human B1 and B3 are from previously published reports.

EXAMPLE II
B~ Associates with ~v And ~F Subunits To determine that the novel B subunit of the present invention is associated with an ~ chain similar to other known integrins, antisera against peptides from the cytoplasmic domain sequence of B6 were prepared. The following amino acid peptides from the cytoplasmic sequence of B6 were prepared and used to immunize rabbits:
RGSTSTFKNVTYKHR (residues 763-777) and YKHREKQKVDLSTDC
(residues 774-788). The antisera were raised in rabbits according to standard procedures known in the art.
Briefly, peptides were chemically coupled to keyhole lympet hemocyanin, and were injected in rabbits in either complete (first injection only) or incomplete Freund's adjuvant as described, for example, in Antibodies: A
Laboratorv Manual, E. Harlow and D. Lowe, eds., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724. Antisera were termed 6830 (to peptides correspondinq to res~dues 763-777) and 6341 (to peptides corresponding to residues 774-788).

The resulting polyclonal antibodies were used to immunoprecipitate detergent lysates from the pancreatic carcinoma cell line FG-2 that had been surface radioiodinated according to procedures well known in the art such as described, for example, in Xajiji et al., EMB0 J 3:673-680 (1989). A complex of two bands was precipitated of respectively 150 kilodaltons (Kd) and 97 Kd in SDS-PAGE under non-reducing conditions. Under reducing conditions, the two bands migrated as a diffused ~ l~J~ 24 band, extending from 130 Kd to 116 Kd. These bands were specific since pre-immune serum did not precipitate any of them and they were not present when the immunoprecipitation was carried out in the presence of the corresponding immunogenic peptide. Furthermore, the same complex of two bands was precipitated by both the 6830 and 6841 antibodies, which were raised against independent peptides from the cytoplasmic sequence deduced from B6 cDNA clones.

To determine which of the two precipitated bands corresponds to B6, a SDS-heat denaturated lysate from surface-radioiodinated FG-2 cells was immunoprecipitated with the 6841 antibody. Only the 97 Kd band was detectable (non-reducing conditions), identifying it as the ~6 band. Under reducing conditions, the apparent molecular weight of this band increased to 116 Kd suggesting the presence of many intra-chain disulfide bonds, which is consistent with the primary structure of B6 and of other integrin B chains.

The other band, of 150 Xd or 130 Kd under non-reducing or reducing conditions, respectively, is likely to be an ~ subunit since it dissociates after SDS-heat denaturation of the lysate, indicating that it is non-covalently associated with the B6 polypeptide.
Furthermore, similar to certain other integrin ~ chains, its molecular weight decreases under reducing conditions by about 20 Kd (130 Kd versus 150 Kd under non-reducing conditions) probably due to a disulfide linked small peptide that dissociates upon reduction.

To identify which ~ chain is associated with ~6' the ~B6 integrin complex was purified by immuno-affinity chromatography on a 6841-protein A sepharose matrix according to procedures well known in the art such as described, for example, in Kajiji et al., EMBO J 3:673-W O 92/12236 ~ PC~r/US91/00236 V

680 (1989). The eluted material was immunoprecipitated with antibodies specific for ~1t ~2~ ~3~ ~5, ~r6 and ~v~
which are known to be expressed in FG-2 cells. Only the anti-~V monoclonal antibody 1~2.19, obtained from David 5 Cherish, Ph.D., Scripps Clinic and Research Foundation, La Jolla, California, reacted with the purified material, which indicates that the ~v is associated with B6 in this pancreatic carcinoma cell line.

To confirm this data, immunodepletion 10 experiments on surface-radioiodinated FG-2 lysates were performed according to methods well known in the art such as described in Kajiji et al., EMBo J 3:673-680 (1989).
The cell lysate was depleted with the 6841 anti-B6 antibody or, in parallel, with a control antiserum, and 15 then immunoprecipitated with the 142.19 anti- rV antibody.
A smaller amount of ~v was present in the illununoprecipitation on the B6 depleted lysate and no 97 Kd B6 band was visible. Instead, a smaller band of about 90 Kd was present. It is hypothesized that this smaller band represents the B5 chain also associated with ~v in these cells. In the control lysate depleted with normal rabbit serum, all three bands, 150 Kd (~v), 97 Kd (B6) and 90 Kd (B5) were present after immunoprecipitation with the anti-~V 142.19 antibody.

Another immunodepletion was carried out using 142.19 antibody as the depleting antibody, or in parallel a mouse monoclonal as a control antibody.
Immunoprecipitations of ~v-depleted lysate with anti-~V
142.19 antibodies did not show the presence of any band, 30 indicating that all ~v-containing integrins had been removed. However, the 6841 anti-B6 antibody still precipitated a complex of two bands, one corresponding to B6, the other with a molecular weight close to that of ~v This ~ chain, however, must differ from ~v since it is 35 unreactive with anti-~V monoclonal antibodies and is W O 92/12236 ~ PC~r/US91tO0236 ~ 26 referred to herein as ~F. In the control depleted lysates, the 6841 anti-B6 antibody precipitates much stronger bands, consistent with the possibility that, in FG-2 cells, two J36 integrins exist, ~VJ36 and ~FJ36.

Although the invention has been described with reference to the presently preferred embodiment, it should be understood that various modifications can be made without departing from the spirit of the invention.
Accordingly, the invention is limited only by the claims.

Claims

WE CLAIM:

1. A substantially purified integrin cell surface receptor subunit comprising .beta.6.

3. A substantially purified integrin comprising .beta.6 bound to an a subunit.

4. The integrin of claim 3, wherein the subunit is .alpha.v.

5. The integrin of claim 3, wherein the subunit is .alpha.p.

6. A substantially purified amino acid fragment specific to .beta.6.

7. A vector comprising a gene encoding for the amino acid fragment of claim 6.

8. A host containing the vector of claim 7.

9. A reagent having specificity for an amino acid sequence specific for .beta.6, wherein said reagent can be used distinguish the .beta.6 subunit from other molecules.

10. The reagent of claim 9, wherein said reagent is an antibody.

11. A substantially purified nucleic acid encoding .beta.6.

13. A substantially purified nucleic acid which specifically hybridizes with the nucleic acid of claim 12 and does not hybridize with a nucleic acid encoding a non-.beta.6 polypeptide.

14. A method of preventing the binding of a cell expressing a .beta.6-containing integrin to ligand capable of binding to said .beta.6-containing integrin, comprising blocking the binding of the .beta.6-containing integrin and the ligand.

15. The method of claim 14, wherein the blocking is effected by binding the .beta.6-containing integrin with a reagent specific thereto.

16. The method of claim 14, wherein the blocking is effected by binding the ligand of the .beta.6-containing integrin with a reagent specific for the ligand.

19. A method of detecting a ligand that binds a .beta.6-containing integrin, comprising contacting the .beta.6-containing integrin with a solution containing the ligand suspected of binding .beta.6-containing integrins and detecting the presence of the ligand bound to the .beta.6-containing integrin.

20. A method of increasing cell adhesion in cells expressing a .beta.6-containing integrin, comprising overexpressing the .beta.6-containing integrin in a cell.

21. A method of decreasing cell adhesion in cells expressing a .beta.6-containing integrin comprising binding the .beta.6-containing integrin with a ligand.